PROJECT SUMMARY/ABSTRACT Signal transduction pathways play pervasive and important roles in normal cellular physiology, and alterations in these pathways are found in many human diseases. One under-studied aspect of many signal transduction pathways is the role of scaffold proteins, which act as integrating platforms for signaling proteins. The NF-?B signaling pathway is altered in many inflammatory and immune diseases and cancers, and is thus viewed as target for therapeutic intervention. The overall project goal is to advance our understanding of the signaling scaffold protein NF-?B essential modulator (NEMO), a component of the inhibitor of ?B kinase (IKK) complex, which is a key regulatory node for NF-?B signaling. In addition to NEMO playing a role in the chronic hyperactivity of NF-?B in human diseases, mutations in NEMO are found in several human immunodeficiency diseases. The long-term goals of the project are to understand how scaffolding proteins such as NEMO use conformational change to regulate the functional interactions between the signaling proteins that are bound to them, to elucidate the structural basis for disease-causing mutations in key regions of NEMO, and to identify new target sites for small molecule drugs that modulate NEMO activity. The following specific aims will be pursued: 1. Structural, biophysical and molecular approaches will be used to characterize the newly discovered ?Intervening Domain? (IVD; aa 111-195) of NEMO, and to elucidate the role that the IVD plays in modulating IKK? binding. Additionally, the mechanism(s) underlying immunodeficiency disease-causing mutations in the IVD region will be established. 2. The recently discovered interaction between NEMO and I?B? will be characterized using biochemical and cellular approaches, to establish whether this interface provides alternative target sites for therapeutic intervention. 3. The mechanism by which NEMO regulates the phosphorylation of I?B by IKK?, and the role in this process played by a conformational change in NEMO, will be assessed and defined. The project team includes three Principal Investigators with expertise and experience across all aspects of the project, including assay development, quantitative and mechanistic biochemistry, X-ray crystallography and small-angle X-ray scattering (SAXS), biophysics, drug discovery, and NF-?B pathway biology and disease. The outcomes of this research will further our understanding of the role of NEMO in normal cellular physiology and in NEMO-related genetic immunodeficiencies, and may lead to the development of new therapeutics for human disease.